Suspension type topical formulations comprising cyclic depsipeptide

ABSTRACT

The invention relates to novel topical pharmaceutical compositions in which the active agent is a cyclic depsipeptide of formula (II) 
                         
and to methods for manufacturing such compositions.

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/477,297, filed Apr. 20, 2011; thecontents of which is incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel topical pharmaceuticalcompositions in which the active agent is a cyclic depsipeptide offormula (II)

and to methods for manufacturing such compositions.

BACKGROUND OF THE INVENTION

The cyclic depsipeptide of formula (II) is useful for the treatment andprevention of inflammatory and/or hyperpoliferative and pruritic skindiseases such as atopic dermatitis, psoriasis, pustular psoriasis,rosacea, keloids, hypertrophic scars, acne, Netherton's syndrome orother pruritic dermatoses such as prurigo nodularis, unspecified itch ofthe elderly as well as other diseases with epithelial barrierdysfunction such as aged skin.

The compound of formula (II) is described in international patentapplication WO2009024527.

It is desirable to identify compositions, and uses of these compositionsthat may improve efficiency, bioavailability, stability and/oracceptance by the patient.

These objectives are achieved by providing a composition as describedherein, by providing the composition for use in diseases, particular forthe treatment of dermatological diseases, as described herein and byproviding a process to produce the composition as described herein.

Further aspects of the invention are disclosed in the specification andindependent claims, preferred embodiments are disclosed in thespecification and the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

The compound of formula (II) presents highly specific difficulties inrelation to topical administration and topical galenic compositions, inparticular, including particular stability problems.

The compound of formula (II) shows only moderate solubility in water andaqueous buffers and low solubility in lipophilic excipients. In polarorganic solvents, good solubility is observed. The compound of formula(II) has a tendency to degradeation in a hydrophilic environment, suchas water and polar organic solvents/co-solvents, and is subject tohydrolysis in the presence of water.

For the treatment and prevention of diseases mentioned above, a specificpenetration and permeation profile is of advantage in order to achievehigh concentration of the cyclic depsipeptide of formula (II) in theskin, while limiting permeation through the skin and thus loweringsystemic exposure. These special requirements necessitate thedevelopment of a non-conventional dosage form.

In accordance with the present invention it has been found that stablepharmaceutical compositions comprising cyclic depsipeptide of formula(II) having suitable penetration and permeation profiles are obtained.Consequently, the risk of occurrence of potential undesirableside-effects and/or active agent decay upon storage is diminished andoverall cost of therapy may be reduced.

Terms used in the specification have the following meanings:

“Active agent” as used herein means a cyclic depsipeptide of formula(II). “Active agent” is also intended to represent amorphous andcrystalline forms such as polymorphs. “Active agent” is also intended torepresent a solvate thereof, a pharmaceutical acceptable salt thereofand its mixtures. “Active agent” is also intended to represent materialexhibiting specific solid state properties such as specific crystalforms and/or milled forms of the “Active agent”, e.g. in micronizedform.

“Solvate” as used herein means a crystal form of a compound whichadditionally contains one or more types of solvent molecules, e.g. ethlyacetate, acetonitrile, water, isopropylacetate, in a stoichiometricallydefined amount. Preferably, solvates contain one type of solventmolecule in the crystal lattice.

As used herein, the term “pharmaceutically acceptable salts” refers tothe nontoxic acid or alkaline earth metal salts of the active agent.These salts can be prepared in situ during the final isolation andpurification of the compounds, or by separately reacting the base oracid functions with a suitable organic or inorganic acid or base,respectively. Representative salts include, but are not limited to, thefollowing: acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemi-sulfate, heptanoate, hexanoate,fumarate, hydro-chloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methane-sulfonate,nicotinate, 2-naphth-alenesulfonate, oxalate, pamoate, pectinate,persulfate, 3-phenylproionate, picrate, pivalate, propionate, succinate,sulfate, tartrate, thiocyanate, p-toluene-sulfonate, and undecanoate.Also, basic nitrogen-containing groups can be quaternized with suchagents as alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides, and iodides; dialkyl sulfates like dimethyl,diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl, and stearyl chlorides, bromides and iodides, aralkylhalides like benzyl and phenethyl bromides, and others. Water oroil-soluble or dispersible products are thereby obtained. Basic additionsalts can be prepared in situ during the final isolation andpurification of the compounds, or separately by reacting carboxylic acidmoieties with a suitable base such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation or withammonia, or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on the alkali and alkaline earth metals, such as sodium,lithium, potassium, calcium, magnesium, aluminum salts and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethyl-amine, trimethylamine,triethylamine, ethylamine, and the like. Other representative organicamines useful for the formation of base addition salts includediethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine,pyridine, picoline, triethanolamine and the like and basic amino acidssuch as arginine, lysine and ornithine.

“Topical pharmaceutical composition” as used herein is known in thefield (e.g. see European Pharmacopoeia, 6.3, 01/2009, 0132) and in thecontext of the present invention particularly refers to a composition ofthe suspension type. Such compositions comprise i) the active agent andii) a matrix. The matrix (also referred to as “base”) containspharmaceutically acceptable excipients and is adapted to a topicalapplication. Further, compositions of the invention may be formulated assemi-solid including gels, patch, foam, tincture, solution, (lip) stick,or spray; each of them in the suspension type. Consequently, viscositiesof the compositions of the invention may vary over a broad range;typically they are semi-solid or liquid, preferably semi-solid.Compositions of the suspension type are characterized in that the activeagent is suspended in the matrix; preferably in the form of a“hydrophobic ointment”.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice, fish, birds and the like. In certain embodiments,the subject is a primate. In yet other embodiments, the subject is ahuman.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context

Throughout this specification and in the claims that follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising” as well as the word “contain”, or variationssuch as “contains” or “containing”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It is further understood that the various embodiments, preferences andranges of this invention, as provided/disclosed in the specification andclaims may be combined with other specified features to provide furtherembodiments.

In a first aspect, the present invention provides a topicalpharmaceutical composition comprising a cyclic depsipeptide of formula(II), a hydrophobic matrix and a consistency agent. It is typically asuspension type composition.

The active agent has a tendency to degrade in hydrophilic environmentsuch as water and polar organic solvents/co-solvents and is subject tohydrolysis in the presence of water.

It was found that topical pharmaceutical compositions comprising acyclic depsipeptide of formula (II), a hydrophobic matrix and aconsistency agent, allow the active agent to be formulated into stablecompositions and allow for suitable penetration and permeation profile;especially in view of the fact that the active agent is suspended in thematrix and thus only a small fraction of molecules is dissolved andavailable for penetration. By the use of a consistency agent it ispossible to increase the level of the active agent to a pharmaceuticallybeneficial level in the skin without skin irritation. However,permeation of the active agent through the skin was very low, resultingin no systemic exposure or very low systemic exposure, thus minimizingthe risk of side effects. Further, these compositions show good physicaland chemical stability. This aspect of the invention shall be explainedin further detail below:

The active agent may be obtained according to the methods described inWO2009024527. Particularly suitable for the inventive compositions areactive agents of the invention in micronized form (×90<20 micrometer).The amount of active agent in the inventive composition may vary over abroad range, it is typically provided in an effective amount. Aneffective amount refers to an amount of the active agent which, whenadministered to a mammal (preferably a human), is sufficient to effect atreatment as defined below. Suitable amounts for the active agent may bedetermined by the skilled person in routine experiments; typically theyare in the range between 0.1-5 wt. %, preferably 0.5-2.0 wt. %, such as0.5, 0.8 or 1.0 wt. %.

Hydrophobic matrix: According to this aspect of the invention, thematrix contains paraffines (hard, liquid, light liquid), vegetable oils,animal fats, synthetic glycerides, waxes, perflourcarbons,semiperflourcarbones and/or liquid polysiloxanes. Typically, thehydrophobic matrix can absorb only small amounts of water. Preferably,the hydrophobic matrix contains one or more types of hydrocarbons;preferably at least two types of hydrocarbons. It was found that suchmatrix disperses a high amount of active agent and produces a stablecomposition. Suitable hydrocarbons are known in the field and may beselected by a skilled person to be compatible with the finalpharmaceutical composition. Suitable hydrocarbons include solid andliquid hydrocarbons which may be linear and/or branched. Suchhydrocarbons are known excipients for pharmaceutical compositions andare commercially available (e.g. as mixtures of individual components).Suitable hydrocarbons include “mineral oil”, “petrolatum”,“microcrystalline wax”. A suitable hydrophobic matrix may contain up to66 wt. % mineral oil, preferably 20-40 wt. % mineral oil. A suitablehydrophobic matrix may contain up to 98 wt. % petrolatum, preferably40-60 wt. % petrolatum. A suitable hydrophobic matrix may contain up to25 wt. % microcrystalline wax, preferably 5-20 wt. % microcrystallinewax. A suitable hydrophobic matrix may contain mineral oil andpetrolatum in a ratio between 1:1 to 1:3, preferably 1:1.5 to 1:2.0.Further, a suitable hydrophobic matrix may contain mineral oil andmicrocrystalline wax in a ratio between 1:0.2 to 1:1, preferably 1:0.33to 1:0.66.

Consistency agent: As used in the context of this invention, agents tomodify consistency, also named consistency improver are known in thefield. Appropriate compounds may be selected by a skilled person to becompatible with the final pharmaceutical composition. It is understoodthat one or more of such agents may be used. Particularly suitable areconsistency agents selected from the group consisting of saturated fattyacids and saturated fatty acid esters. Preferred are saturated C6-C30fatty acids, -esters; particularly preferred are C10-C20 fatty acids,-esters. Further, linear fatty acids, -esters are preferred. For esters,C1-C4 alkyl groups are preferred. Among these consistency agents,isopropyl myristate is particularly suitable. The amount of consistencyagent in the inventive composition may vary over a broad range, it istypically provided in an effective amount. Suitable amounts ofconsistency agent may be determined by the skilled person in routineexperiments; typically they are between 2.5-20 wt. %, preferably 2.5-10wt. % of the total composition.

In one embodiment, the invention relates to a composition according tothis aspect of the invention which contains no further excipients. Thus,the inventive composition only contains (i.e. consist of or essentiallyconsists of) the active agent, one or more hydrocarbons and aconsistency agent. Such compositions are considered advantageous e.g.for simple manufacturing and/or for patient populations with increasedskin irritation/allergic potential towards other excipients.

In a further embodiment, the invention relates to a compositionaccording to this aspect of the invention which contains one or moreadditional excipients. Such excipients are known in the field and may bereadily identified by the skilled person. Suitable excipients may beselected from the group consisting of antioxidants, gelling agents, phadjusting agents/buffers, penetration enhancers, preservatives,(co-)solvents and stabilizers. Such excipients are known in the field,commercially available and may be identified in standard textbooks, suchas the Handbook of Pharmaceutical Excipients by R. C. Rowe et al. Suchcompositions are advantageous to specifically adapt to manufacturers orpatients needs and thus improve product properties (like shelf life orpatient compliance). Suitable further excipients are explained below:

Antioxidants are known in the field and may be selected by a skilledperson to be compatible with the final pharmaceutical composition. It isunderstood that one or more antioxidants may be used. It was found thatthe antioxidant stabilizes the agent of the invention. Preferably, theantioxidant is selected from the group consisting of phenole derivatives(e.g. butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA));ascorbic acid derivatives (e.g. ascorbic acid, ascorbyl palmiate),tocopherol derivatives (e. g. Vitamin E, Vitamin E TPGS), bisulfitederivatives (Na bisulfite, Na meta bisulfite) and thio urea. Morepreferably, is selected from the group consisting of butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), alpha tocopherol,ascorbic acid or a mixture of thereof. Particularly preferably, theantioxidant is BHT. A suitable composition may contain up to 2 wt. %antioxidant, preferably 0.005-0.5 wt. %.

Gelling agents are known in the field and may be selected by a skilledperson to be compatible with the final pharmaceutical composition. It isunderstood that one or more gelling agents may be used. Gelling agentsare included in the compositions of this invention to adjust viscosity.Gelling agents that are suitable for lipophilic formulations, e.g.aerosil, polyethylen, and aluminum soap. A suitable composition maycontain up to 10 wt. % gelling agent, preferably 0.02 to 2 wt. %.

Agents to adjust the pH or to provide a pH buffer are known in thefield. Appropriate buffers may be selected by a skilled person to becompatible with the final pharmaceutical composition. It is understoodthat one or more of such buffers may be used. A suitable composition maycontain such buffers to adjust the pH of the inventive composition inthe range of 4-8, preferably 5-7, such as 6.5.

Penetration enhancers are known in the field and may be selected by askilled person to be compatible with the final pharmaceuticalcomposition. It is understood that one or more preservatives may beused. As used herein, the term “penetration enhancer” refers to asubstance that enhances, i.e. improves, the penetration of the activeagent when administered topically, (epicutanously), to the skin ormucosa, e.g. to skin. A wide range of penetration enhancers may be used.Suitable are penetration enhancers can for example be selected from thegroup consisting of:

-   -   alcohols such as ethanol, 2-propanol, propylene glycol, ° leyl        alcohol, linolenyl alcohol;    -   fatty acid ester such as butyl acetate, glycerol mono laureate,        diethylene glycol oleate,    -   fatty acids such as oleic acid;    -   saponins    -   amines such as urea, N,N-diethyl-m-toluamide;    -   surfactants such as Brij 36T, PLURONIC® F68 block (copolymers        based on ethylene oxide and propylene oxide),    -   others such as terpenes, dimethyl sulfoxide,        1,3-dioxacyloalkanes (SEPA), azone, diethylene glycol monoethyl        ether, dimethylispropyladipate, dimethyl-isosorbid.

The amount of penetration enhancer in the inventive composition may varyover a broad range, it is typically provided in an effective amount.Higher penetration may also result in an increased permeation, e.g.increased permeation through the skin. Preferably, the delivery of theactive agent to the systemic circulation is not or not significantlyenhanced (no or no significant permeation). Suitable amounts ofpenetration enhancer may be determined by the skilled person in routineexperiments; typically they are between 2.5-20 wt. %, preferably 2.5-10wt. % of the total composition.

Preservatives are known in the field and may be selected by a skilledperson to be compatible with the final pharmaceutical composition. It isunderstood that one or more preservatives may be used. Preservatives areincluded in the pharmaceutical compositions of this invention toincrease shelf life. Preferably, preservatives are selected from thegroup of alcohols (e.g. benzyl alcohol), phenols, andparahydroxybenzoates. More preferably, preservatives are selected fromparabens, alcohols, biguanides, mercuric salts, imidurea. A suitablecomposition may contain up to 5 wt. %, preferably 0.01 to 3 wt. %.

Co-solvents and solvents are known in the field and may be selected by askilled person to be compatible with the final pharmaceuticalcomposition; it denotes an excipient which dissolves the agent of theinvention (partly or fully) and has a high miscibility with water. Asolvent is an excipient which dissolves the agent of the invention buthas a low miscibility with water. Thus, depending on the type ofcomposition and the other excipients present, a specific compound mayserve as a solvent or as a co-solvent. It is understood that one or moreco-solvents/solvents may be used.

The active agent may be prepared as described in international patentapplication WO2009024527. They may include other solvents, for examplesolvents which may have been used for the purification or, as mentionedtherein, in form of salts.

In accordance with the present invention the active agent may be presentin an amount by weight of up to about 20% by weight of the compositionof the invention, e.g. from about 0.05% by weight. The active agent ispreferably present in an amount of 0.5 to 5% by weight of thecomposition, more preferably in an amount of 0.2 to 1% by weight of thecomposition.

The invention relates in a second aspect to a method for manufacturingcompositions as described herein comprising the step of combining theexcipients as described herein to obtain a hydrophobic matrix, combiningthe thus obtained matrix with the active agent.

A composition according to this invention may be prepared by processesthat are known per se, but not yet applied for the present compositionswhere they thus form new processes. In general, the manufacture of apharmaceutical composition utilizes standard pharmaceutical processescomprising the step of combining the active agent with a matrix, e.g. bymixing, dissolving and/or lyophilizing. Such steps may also compriseheating or cooling the materials used. As outlined above, the activeagent is available according to known processes; the individualcomponents of the matrix are either known per se or available accordingto known processes.

In one embodiment, the invention relates to a method of manufacturing acomposition as described in the first aspect of the invention (i.e. acomposition of the suspension type) comprising the steps of

-   -   combining all excipients at a temperature between 30-95° C. to        obtain a melt,    -   adding the active agent, preferably at a temperature between        30-95° C., to obtain a suspension,    -   homogenizing the obtained composition.    -   optionally cooling down the obtained composition.

The invention is illustrated by the following Examples.

Abbreviations: ° C. degree(s) Celsius rpm revolutions per minute wt. %or % by weight. weight percent MBq mega Becquerel RH relative humidity

EXAMPLES

1. Pharmaceutical Compositions

An ointment, suspension type, was prepared by combining the excipientsas indicated below with the compound of formula II. Specifically, allcomponents as indicated below, except for the compound of formula II,were combined and heated to 80° C. with stirring to obtain a melt. Thecompound of formula II was added at this temperature and the resultingmixture was stirred until a complete wetting of the compound of formulaII was obtained. The suspension was then homogenized with an ULTRATURRAX: homogenizer (from company named IKA), at 24000 rpm for 5 min at80° C. The obtained composition was slowly cooled down to 25° C. toobtain a composition of the suspension type.

ointment ointment ointment ointment Var A [%] Var B [%] Var C [%] Var D[%] Compound of formula II 0.5 1.0 0.2 0.1 White VASELINE 54 53.5 54.354.4 (petrolatum) liquid paraffin (mineral 30 30 30 30 oil)microcrystalline wax 12.5 12.5 12.5 12.5 (hydrocarbons) Isopropylmyristate 3.0 3.0 3.0 3.0

2. Stability Tests

2a) Chemical Stability

The pharmaceutical compositions, as prepared above, were tested forchemical stability. After 5 months of storage at 5° C., room temperatureand 40° C., less than 0.1% degradation product is detected. After 12months of storage at 5° C., 25° C./65% RH and 30° C./65% RH, and after 6month at 40° C. less than 0.1% degradation product is detected. Thechemical stability of the compositions was found to be satisfying atlong term conditions of 5° C. over 12 months and at acceleratedconditions of 25° C./60% RH over 6 months. The chemical stability of thecompositions was found to be very good.

2b) Physical Stability

The pharmaceutical compositions, as prepared above, were tested forphysical stability. After 5 months of storage at 5° C., room temperatureand 40° C., suitable particle size distribution was found at all storageconditions. After 12 months of storage at 5° C., 25° C./65% RH and 30°C./65% RH, and after 6 month at 40° C. suitable particle sizedistribution was found at all storage conditions. The physical stabilityof the compositions was found to be satisfying at long term conditionsof 5° C. over 12 months and at accelerated conditions of 25° C./60% RHover 6 months. The physical stability of the compositions was found tobe very good.

2c) Temperature Cycling Test

The pharmaceutical compositions, Var B and C, were tested in atemperature cycling test.

The samples were cycled between 40° C. and 5° C. in 24-hour intervalsfor one month.

Afterwards the physical characteristics of the samples were analyzed.

No changes of the visual and microscopic appearance were observed. Astrong increase of the viscosity of the ointment could be observed.

2d) Freeze and Thawing Test

The pharmaceutical compositions, Var B and C, were tested in a freezeand thawing test.

The samples were stored for four complete freeze thaw cycles of −20° C.for 6 days, followed by 1 day at 25° C./60% RH. Samples were taken after28 days and the physical characteristics of the samples were analyzed.No changes of the visual and microscopic appearance were observed andthe viscosity of the ointment did not change.

2e) In-Use Test

The pharmaceutical composition, Var B was tested in an in-use test.

The sample was placed in white aluminum tubes (10 g) with internalprotective lacquer, without imprint, with membrane, equipped with awhite screw cap with a built-in point.

Approximately 0.1 g ointment was removed from the 10 g tube twice daily(morning and late afternoon) for 7 and 14 working days. After eachremoval of ointment, the tubes were tightly closed and stored at 25° C.until the next removal.

After an in-use period of 7 days and 14 days at 25°, the assay of thecompound of formula (II) remained unchanged.

3. Allergic Contact Dermatitis (ACD) in Domestic Pigs:

For sensitization 500 μl of 10% 2,4-dinitrofluorobenzene (DNFB,dissolved in DMSO: acetone: olive oil [1:5:3, v/v/v]) were appliedepicutaneously in divided volumes onto the inner lateral aspects and tothe basis of both ears and onto both groins. One week later, cutaneoushypersensitivity reactions were elicited with 15 μl 1% DNFB at 12-16sites on the shaved dorsolateral back. The test sites, 7 cm² in size,were arranged craniocaudally on both dorsal sides. Fifty microlitersamples of formulations were applied to 2 test sites each on the rightsides, 0.5 and 6 hrs after the challenge on day 8. The contralateralleft sites were similarly treated with the vehicle (placebo) alone. Thetest sites were clinically examined 24 hrs after the challenge wheninflammation peaked. The changes were scored on a scale from 0 to 4(Table 3-1), allowing a combined maximal score of 12 per designatedsite.

TABLE 3-1 Scoring of clinical signs of test sites affected with ACDScore Erythema/Intensity Erythema/Extent Induration 0 absent absentabsent 1 scarcely visible small spotted scarcely palpable 2 mild largespotted mild hardening 3 pronounced confluent pronounced hardening 4severe (or livid homogenous redness pronounced and discoloring) of testsite elevated hardening of test site

The results are summarized in Table 3-2.

TABLE 3-2 Inhibition of clinical ACD score by compound preparations %inhibition compared to Statistical Preparation vehicle-treated controlssignificance 0.5% cream + linoleic acid* 22 p < 0.01 0.5% cream −linoleic acid** 13 ns 0.5% ointment Var A 16 p < 0.05 1.0% ointment VarB 38 p < 0.001 1.0% in propylene 33 p < 0.01 glycol/ethanol 7/3 (v/v)*0.5% cream + linoleic acid - components: compound of formula II: 0.50%,glycerin anhydrous: 64.45%, Miglyol 812 (triglyceride mikett): 25.00%,Sedefos 75: 9.00%, butylhydroxytoluene: 0.05%, linoleic acid: 1.00%**0.5% cream − linoleic acid - components: compound of formula II:0.50%, glycerin anhydrous: 65.45%, Miglyol 812 (triglyceride mikett):25.00%, Sedefos 75: 9.00%, butylhydroxytoluene: 0.05%

4. In vivo Skin Penetration/Permeability Study of the Suspension TypeFormulation in Pigs

In order to determine the flux (penetration) of the epicutaneouslyapplied compound into the dermis under in vivo conditions, 4 cm² sizedareas on the dorsolateral back of pigs were treated with compoundformulations 8, 4, 2, and 1 hrs prior to dissection. Epidermis wasremoved from the excised skin after heat separation, and 6 mm punchsamples of dermal sheets of 1 mm thickness from the de-epidermized skinwere analysed for compound concentrations. This procedure enabledreliable determinations of drug levels in the dermis without the risk ofcontamination of the analytes with residual non-absorbed drug present onthe treated skin surface or trapped in the superficial stratum corneum.

The drug levels obtained in the dermis are listed in Table 4-1.

TABLE 4-1 Drug levels in pig dermis after topical application in vivoointment Var B (1.0%) ointment Var A (0.5%) Mean conc. Mean conc. Time[hr] (μg/g) SEM (n) (μg/g) SEM (n) 1 0.399 0.102 (8) 0.077 0.025 2 0.4960.209 (8) 0.216 0.069 4 0.199 0.049 (8) 0.284 0.126 8 0.426 0.177 (8)0.281 0.062

The concentrations reached in the skin after the application of 1% and0.5% ointment over 1-8 hours were comparable to the levels reached inthe in vitro skin penetration assay in whole skin after 48 hours. (seebelow).

Penetration into Stratum Corneum of Pig Skin in vivo

Lateral thighs of domestic pigs were treated topically with ointment VarB (1.0%) 2 hrs and, contralaterally 0.5 hr prior to the dissection ofthe treated skin samples. Excess of applied material was removed bywiping with a paper towel. D SQUAME® tapes (adhesive tape for skinsampling, 2.2 cm in diameter, CuDerm) were used to serially remove 40serial layers of stratum corneum using and air pressure-driven plungerto obtain uniform pressure. Compound levels were analyzed and normalizedto the skin area stripped.

Compound levels in the stratum corneum (sum of 40 tape strip extracts)amounted to a total of 3.6 μg/m² and 7.5 μg/cm² 0.5 and 2 hours afterapplication of the ointment Var B (1.0%), respectively.

5. In vitro Skin Penetration/Permeation in Human Abdominal Cadaver Skin

Skin Preparation

Frozen human abdominal cadaver skin was obtained from the West HungarianRegional Tissue Bank, Györ, Hungary (Batch No. 090620-9 (=batch 1) and090609-8 (=batch 2), from a 69 and a 61-years old female donor,respectively. Before starting the experiment, the skin was kept at −20°C. for approximately 4 months. Thawed skin samples were dermatomized toa thickness of 500 μm with an AESCULAP dermatome (Aesculap AG,Tuttlingen, Germany), cut to fit into the diffusion area, and assembledbetween the donor and the receptor chambers of the diffusion Franz cells(Franz 1975).

Determination of the Skin Integrity

The integrity of skin was determined by evaluating the permeation oftritiated water (³H₂O) through the skin; 400 μL of ³H₂O (0.1 MBq/mL)were applied to the surface of the skin. After 30 min of equilibration,the ³H₂O was removed from the skin with cotton tips; 2 mL of thereceptor phase (composition of the receptor fluid described below) weresampled in order to measure the fraction of ³H₂O which permeated acrossthe skin. Radioactivity in the receptor phase was measured by liquidscintillation counting in Liquid Scintillation Systems 2500 TR (PackardInstr. Co., Meriden, Conn., USA). For quench correction an externalstandard method was used. Quench correction curves were established bymeans of sealed standards (Packard Instr.)

Determination of the in vitro Skin Permeation Through the Skin andPenetration into the Skin

The skin was used as a membrane separating the donor and receptorchambers of the manual static Franz cells (volume of approximately 7.3mL, 16 mm internal diameter). The receptor chamber was filled with thereceptor fluid (0.5% aqueous solution of Brij 78 [Volpo20]) to simulatethe human physiological conditions and the systemic removal of the drugfrom skin. The receptor fluid contained in addition 100 U/mL of a 1%penicillin/streptomycin mixture to prevent microbiologicalcontamination.

The effective skin areas for diffusion and the volumes of receivercompartment were in the range of 1.78 to 2.14 cm² (mean 2.01 cm²) and6.98 to 7.54 mL, respectively. The temperature of the cells was keptconstant using a circulating water bath at 34±1° C. Magnetic stirrerbars were constantly used during the entire experiment to ensurereceptor uniformity.

Collection and Handling of the Samples

The formulations a-c (Table 5) in amounts of 243-305 mg and 0.300 mL offormulation d were applied as a single dose on the skin samples mountedon the diffusion Franz cells (corresponding to sampling time=0 h, 3-4cells per formulation). The formulations were left on the skin for 48 h.To avoid evaporation and dryness of the formulations, the donorcompartments of Franz cells were semi-occluded with parafilm (Parafilm®M) containing holes. For determination of active agent that permeatedacross the skin, aliquots of 200 μL of the receptor fluid were collectedfrom the receptor compartment at 4, 7, 20, 25.5, 28, 31, 44, and 48 hafter application. The volume taken from the receptor compartment wasreplaced every time with the same volume of fresh receptor fluid inorder to keep the total receptor fluid volume constant during the entireassay period.

At the end of the treatment period (48 h post application), the residualformulation on the surface of each skin sample was carefully removedwith a cotton tip applicator, and the application area was washed with acotton containing water and gently dried with new cotton applicators.The procedure was repeated three times. The stratum corneum was thenseparated from the skin by 21 tape strips using a commercial adhesivetape (Scotch® 550, 3M). The first strip was discarded in order to avoidpotential contamination from the formulation and the remaining 20 stripswere placed into vials (strips no. 2-6, 7-11, 12-16, 17-21 pooledtogether). Biopsies of the treated area of the stripped skin were takenwith a 1.2 cm diameter punch and weighed. Receptor fluids, tape strips,and stripped skins samples were frozen and kept at −20° C. untilanalysis. The concentration of the cyclic depsipeptide of formula (II)in samples was determined by a validated LC-MS/MS analysis; the lowerlimit of quantification was 0.5 ng/mL (receptor fluid and strips) or 5ng/g (skin samples).

TABLE 5 Results (mean ± SD [range], n = 1-4) 1% in propyleneFormulation/cyclic depsipeptide ointment Var D ointment Var A ointmentVar B glycol/ethanol of formula (II) (0.1%) (0.5%) (1.0%) 7/3 (v/v)Concentration in the stratum 3270  5330 ± 3730 4920 ± 3180  50.3 ± 62.1corneum: 2-21 strips (ng/cm²)  [2730-3810] [2090-10600]  [1250-6680][0.00-120] Skin concentration after 48 h  2.17 ± 3.68  11.4 ± 15.1  20.8± 32.4  185 ± 196 (ng/cm²) [0.187-7.69] [1.02-33.7] [0.479-68.5][1.32-391] 0.330 ± 0.013^(a)  3.90 ± 0.259^(a)  4.91 ± 0.749^(a) Skinconcentration after 48 h  48.1 ± 83.3   204 ± 300  370 ± 606 3520 ± 3620(ng/g)  [5.08-173] [16.9-652]  [9.63-1270] [39.4-7260]  6.40 ± 0.144^(a) 54.7 ± 3.36^(a)  70.1 ± 10.3^(a) Flux [range] (ng/cm²/h) 0.825 ± 1.650.970 ± 1.94  4.63 ± 9.26  25.0 ± 26.0 [0.00-3.30] [0.00-3.88] [0.00-18.5] [0.00-51.8]  0.00 ± 0.00^(a)  0.00 ± 0.00^(a)  0.00 ±0.00^(a) Lag time (h) 0.422 15.4 13.2 17.9 NC^(a) NC^(a) NC^(a)^(a)highest value (outlier) excluded, SD calculated for n > 2

What is claimed is:
 1. A topical pharmaceutical composition ofsuspension type comprising a cyclic depsipeptide of formula (II)

a hydrophobic matrix and isopropyl myristate as a consistency agent. 2.A pharmaceutical composition according to claim 1, in form of ahydrophobic ointment.
 3. A pharmaceutical composition according to claim1, wherein the hydrophobic matrix includes one or more compoundsselected from the group consisting of paraffines, vegetable oils, animalfats, synthetic glycerides, waxes, perflourcarbons, semiperflourcarbonesand liquid polysiloxanes.
 4. A composition according to claim 1, whereinthe hydrophobic matrix includes at least two types of hydrocarbonsselected from the group consisting of mineral oil, petrolatum, andmicrocrystalline wax.
 5. A composition according to claim 1 furthercomprising one or more excipients selected from the group consisting of:a. an antioxidant which is butylated hydroxytoluene, b. gelling agentsselected from the group consisting of colloidal silicon dioxide,polyethylen, and aluminum soap, c. ph adjusting agents/buffers, d.penetration enhancers selected from the group consisting of ethanol,2-propanol, propylene glycol, oleyl alcohol, linolenyl alcohol, butylacetate, glycerol mono laureate, diethylene glycol oleate, oleic acid,saponins, urea and N,N-diethyl-m-toluamide; and e. preservativesselected from the group consisting of parabens, alcohols, biguanides,mercuric salts, imidurea.
 6. A composition according to claim 1 furthercomprising one or more penetration enhancer selected from the groupconsisting of ethanol, 2-propanol, propylene glycol, oleyl alcohol,linolenyl alcohol, butyl acetate, glycerol mono laureate, diethyleneglycol oleate, oleic acid, saponins, urea and N,N-diethyl-m-toluamide.7. A composition according to claim 1, wherein the cyclic depsipeptideof formula (II) is present in an amount between 0.1-5 wt. % of the totalcomposition, the consistency agent is present in an amount between2.5-20 wt. % of the total composition and the hydrophobic matrixcontains up to 66 wt. % mineral oil, up to 98 wt. % petrolatum, up to 25wt. % microcrystalline wax.
 8. A pharmaceutical composition according toclaim 2, wherein the hydrophobic matrix includes one or more compoundsselected from the group consisting of paraffines, vegetable oils, animalfats, synthetic glycerides, waxes, perflourcarbons, semiperflourcarbonesand liquid polysiloxanes.
 9. A composition according to claim 2, whereinthe hydrophobic matrix includes at least two types of hydrocarbonsselected from the group consisting of mineral oil, petrolatum, andmicrocrystalline wax.
 10. A composition according to claim 3, whereinthe hydrophobic matrix includes at least two types of hydrocarbonsselected from the group consisting of mineral oil, petrolatum, andmicrocrystalline wax.
 11. A composition according to claim 2, whereinthe cyclic depsipeptide of formula (II) is present in an amount between0.1-5 wt. % of the total composition, the consistency agent is presentin an amount between 2.5-20 wt. % of the total composition and thehydrophobic matrix contains up to 66 wt. % mineral oil, up to 98 wt. %petrolatum, up to 25 wt. % microcrystalline wax.
 12. A compositionaccording to claim 3, wherein the cyclic depsipeptide of formula (II) ispresent in an amount between 0.1-5 wt. % of the total composition, theconsistency agent is present in an amount between 2.5-20 wt. % of thetotal composition and the hydrophobic matrix contains up to 66 wt. %mineral oil, up to 98 wt. % petrolatum, up to 25 wt. % microcrystallinewax.
 13. A composition according to claim 4, wherein the cyclicdepsipeptide of formula (II) is present in an amount between 0.1-5 wt. %of the total composition, the consistency agent is present in an amountbetween 2.5-20 wt. % of the total composition and the hydrophobic matrixcontains up to 66 wt. % mineral oil, up to 98 wt. % petrolatum, up to 25wt. % microcrystalline wax.
 14. A composition according to claim 5,wherein the cyclic depsipeptide of formula (II) is present in an amountbetween 0.1-5 wt. % of the total composition, the consistency agent ispresent in an amount between 2.5-20 wt. % of the total composition andthe hydrophobic matrix contains up to 66 wt. % mineral oil, up to 98 wt.% petrolatum, up to 25 wt. % microcrystalline wax.